Recent developments in the pharmaceutical industry and combinatorial chemistry have exponentially increased the number of potentially useful compounds, each of which must be characterized (i.e., the components and structure must be understood) in order identify the active components and/or establish processes for synthesizing the compounds. In an effort to increase the speed with which these compounds are analyzed, researchers have sought to introduce a higher degree of automation in the analytical process as well as increase the number of analyses performed in parallel.
One useful analytical process is chromatography, which encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. Liquid chromatography (“LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) disposed within a tube (or other channel boundary). The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with a minimal void between each particle, since better resolution during use is typically obtained from more tightly packed columns. As an alternative to packed particulate material, a porous monolith or similar matrix may be used. So-called “high performance liquid chromatography” (“HPLC”) refers to efficient separation methods that are typically performed at high operating pressures.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Examples of types of stationary phases to support such interactions are solids, ionic groups on a resin, liquids on an inert solid support, and porous or semi-porous inert particles, respectively. Commonly employed base materials include silica, alumina, zirconium, or polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform other (e.g., adsorption or ion exchange separation) techniques.
Mobile phase is forced through the stationary phase using means such as, for example, one or more pumps, gravity, voltage-driven electrokinetic flow, or other established means for generating a pressure differential. After sample is injected into the mobile phase, such as with a conventional loop valve, components of the sample will migrate according to interactions with the stationary phase and the flow of such components are retarded to varying degrees. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a component to emerge from the column with the mobile phase. In other words, as the sample travels through voids or pores in the stationary phase, the sample may be separated into its constituent species due to the attraction of the species to the stationary phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following separation in an LC column, the eluate stream contains series of regions having an elevated concentration of individual component species. Thus, HPLC acts to provide relatively pure and discrete samples of each of the components of a compound. Gradient separations using conventional HPLC systems are typically performed within intervals of roughly five to ten minutes, followed by a flush or rinse cycle before another sample is separated in the same separation column.
Following chromatographic separation in the column, the resulting eluate stream (consisting of mobile phase and sample) contains a series of regions having elevated concentrations of individual species, which can be detected by various flow-through techniques including spectrophotometric (e.g., UV-Vis), fluorimetric, refractive index, electrochemical, or radioactivity detection. Liquid chromatography with flow-through detection generally provides signal response that is proportional to analyte amount or concentration.
Because liquid chromatography is useful in separating, identifying, purifying, and quantifying compounds within various mixtures, it would be desirable to perform multiple chromatographic separations simultaneously. By increasing the number of separations performed in parallel, researchers and scientists may increase the rate at which compounds of interest are isolated and characterized. Nonetheless, the ability to perform multiple parallel separations has been limited for a variety of reasons.
Typically, separation columns used for high performance liquid chromatography (HPLC) are contained in tubes made from high strength materials (to withstand the high pressures that are required by the separation process). Such a tube contains packed particulate stationary phase material, which is retained within the tube by porous “frits” (typically, small coin-shaped pieces of sintered metal or silica) positioned at both ends of the tube. The frits (and the stationary phase) are retained by ferrules and nuts or other appropriate fasteners. One drawback of this type of separation column is the complex and time-consuming fabrication process required to assemble the column.
Another drawback to using conventional separation columns for high-throughput applications is that they must interface with other components of the system through complex fittings, frequently requiring input and output lines to be screwed on to the column. As a consequence of these complex configurations, performing multiple separations in parallel becomes increasingly complex with the addition of each new column to be operated. Moreover, automation of such devices also is challenging, requiring automated systems capable of performing complex tasks such as precisely aligning components and rotating screw fittings.
For example, PCT Patent Application No. WO 02/28509 by Strand et al. (“Strand”) discloses a fluid separation conduit cartridge that includes a conventional separation column curved into a “U” shape and encased in a cartridge housing. While the cartridge according to Strand appears to avoid the need for threaded connectors between the cartridge and the instrument, its use of conventional separation column technology still requires complex fabrication operations and numerous parts, such as the ferrule sub-assemblies illustrated in FIGS. 4-7 therein. Moreover, the U-shaped separation column may be undesirable, as fluid flowing along one side of the separation column will have a different path length than fluid flowing along the other side, which may result in peak spreading or other inaccuracies in analytical results. Finally, the cartridge disclosed in Strand includes only one separation column. Presumably, multiple such cartridges could be used on one instrument; however, the removal and replacement of a large number of cartridges, together with management of cartridge re-use, would be time consuming and complex. Presumably, multiple U-shaped separation columns could be included in one cartridge; however, given the nature of this separation column (i.e., conventional design with threaded fitting to retain the stationary phase material), such a cartridge would be large and bulky, eliminating one of the principal advantages of microfluidic operations—small instrument footprint.
In another example, PCT Patent Application No. WO 01/09598 by Holl et al. (“Holl”) discloses a manifold for providing fluids to a microfluidic de-gassing device. The manifold according to Holl clamps onto a microfluidic device, pressing the protruding end of an interconnect tube into an elastomeric layer on the surface of and surrounding the inlet orifice of the microfluidic device. The system disclosed therein has the disadvantage of requiring an elastomeric layer to be added to the surface of the microfluidic device, the bonding of which layer may be problematic depending on the materials used for the elastomeric layer and the device. For instance, if an adhesive is used to bond the elastomeric layer to the surface of this device, the adhesive may contaminate the samples introduced into the microfluidic device. Adhesiveless bonding methods may limit the materials that may be used for the microfluidic device and elastomeric layer, potentially limiting the types of compatible fluids that may be used in the device. Also, if the elastomeric layer should be damaged, the entire microfluidic device must be repaired or replaced, potential interrupting operations. Moreover, the interconnect system disclosed by Holl requires precise placement of the interconnect tube with respect to the surface of the manifold to ensure that the interconnect tube protrudes a limited distance into the elastomeric layer. This is particularly important as Holl notes that excessive intrusion of an interconnect tube into an elastomeric layer may cause the elastomeric layer to bulge inward into the orifice, thereby occluding the orifice and preventing the flow of fluid through the interface. Finally, the interface disclosed by Holl is intended for use in degassing—an application typically not involving pressures significantly greater than about 50 psi. In contrast, pressures associated with liquid chromatography and HPLC in particular, typically exceed about 100 psi, often exceeding about 200 psi, about 300 psi and even as much as about 500 psi.
Thus, it would be desirable to provide a system for performing multiple liquid chromatography separations in parallel where multiple separation columns may be easily installed within and operated with the system. It also would be desirable to provide a microfluidic interface capable of maintaining a seal at the high operating pressures typically associated with liquid chromatography.
None of the figures are drawn to scale unless indicated otherwise. The size of one figure relative to another is not intended to be limiting, since certain figures and/or features may be expanded to promote clarity in the description.